Methods to screen peptide libraries using minicell display

ABSTRACT

A minicell display method has been developed which has significant advantages for screening peptide libraries for candidates that can bind and effectively modulate a particular biological process. The method, based on the small, anucleate minicell, has increased versatility in generating unique sequences to screen as well as increasing the size of the peptides to be screened. In vivo mutagenesis, at the level of protein synthesis, as well as DNA replication, increases diversification of the library to be screened and therefore substantially increases the number of potential peptides that can modulate a particular biological response or mechanism.

CROSS REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application Ser. No. 60/274,039filed on Mar. 7, 2001, and U.S. Provisional Application Ser. No.60/306,946 filed on Jul. 20, 2001.

FIELD OF THE INVENTION

The present invention is generally in the field of high throughputpeptide screening, and in particular relates to a minicell displaytechnology for generation and screening of random peptides.

BACKGROUND OF THE INVENTION

The interaction between cognate proteins in receptor-ligand complexes,enzyme substrate reactions and antibody-antigen binding reactions hasfurthered the understanding of the molecular interactions required toeffect a response in a wide range of processes. The search for newpeptide molecules which can bind to selected targets and effectivelymodulate a particular biological process is at the forefront ofagricultural, biological, and medicinal research.

There are several examples of methods that use peptides or nucleotidesto develop libraries of potential receptor, enzyme, or antibodyinteracting peptides. Over the course of the last two decades theselibraries have been incorporated into systems that allow the expressionof random peptides on the surface of different phage or bacteria. Manypublications have reported the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedtarget. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378–6382 (1990); Devlin et al., Science 249, 404–406 (1990), Scott &Smith, Science 249, 386–388 (1990); U.S. Pat. No. 5,571,698 to Ladner etal. A basic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the target polypeptide. This physical association is provided by thephage particle, which displays a polypeptide as part of a capsidenclosing the phage genome which encodes the polypeptide. Theestablishment of a physical association between polypeptides and theirgenetic material allows simultaneous mass screening of very largenumbers of phage bearing different polypeptides. Phage displaying apolypeptide with affinity to a target bind to the target and these phageare enriched by affinity screening to the target. The identity ofpolypeptides displayed from these phage can be determined from theirrespective genomes. Using these methods a polypeptide identified ashaving a binding affinity for a desired target can then be synthesizedin bulk by conventional means.

In addition to providing a method for selecting peptides that interactwith target molecules, phage display has been used to direct filamentousphage to target cells using peptides, genetically fused to phage coatproteins, that bind integrin proteins on the surface of mammalian cells.This method of phage display has had a profound influence on genetherapy applications and their attempts to target cells in a specificmanner.

Another approach to obtaining surface expressed foreign proteins hasbeen the use of bacterial native membrane proteins as carriers forforeign protein. In general, many attempts to develop methods ofanchoring proteins on a bacterial surface have focused on fusion of thedesired recombinant polypeptide to a native protein that is normallyexposed on the cell's exterior with the hope that the resulting hybridwill also be localized on the surface. However, in most cases, theforeign protein interferes with localization, and thus, the fusionprotein is unable to reach the cell surface. These fusions either end upat incorrect cellular locations or become anchored in the membrane witha secreted protein domain facing the periplasm. See Murphy, et al., J.Bacteriol., 172:2736 (1990).

Recent advances in bacterial display methods have circumvented thisproblem by using fusion proteins comprising pilin protein (TraA) or aportion thereof and a heterologous polypeptide displaying the librarypeptide on the outer surface of the bacterial host cell capable offorming pilus. See U.S. Pat. No. 5,516,637 to Huang et al. The pilus isanchored to the cell surface of the bacteria and is naturally solventexposed.

Alternatively, the FLITRX™ (Invitrogen Corp.) random peptide libraryuses the bacterial flagellar protein, FliC, and thioredoxin, TrxA, todisplay a random peptide library of dodecamers on the surface of E.coliin a conformationally constrained manner. See Lu et al., BioTechnology,13:366 (1995). These systems have been applied to antibody epitopemapping, the development and construction of live bacterial vaccinedelivery systems, and the generation of whole-cell bio-adsorbants forenvironmental clean-up purposes and diagnostics. Peptide sequences thatbind to tumor specific targets on tumor derived epithelial cells havealso been identified using the FLITRX™ system. See Brown et al., Annalsof Surgical Oncology, 7(10):743 (2000).

Although the phage and bacterial display systems have provided uniqueroutes to elucidating new peptides which can bind target molecules withnew or enhanced binding properties, there are several importantlimitations that need to be considered. Minimal changes in thestructural conformation of the phage coat protein to which the peptideis genetically fused are tolerable. Problems arise when larger peptideinserts (more than 100 amino acids) disrupt the function of the coatprotein and therefore phage assembly. Heterologous peptides have beendisplayed on bacteria using both fimbriae as well as flagellarfilaments. Insert size constraints affect the applicability of thesesystems as well. To date, the largest peptides to be displayed infimbriae range from 50 to 60 amino acids, while the functionalexpression of adhesive peptides fused to the FliC flagellin ofEscherichia coli appears to be restricted to 302 amino acids. SeeWesterlund-Wikstrom 2000.

Amino acid analogs have been used to replace chemically reactiveresidues and improve the stability of the synthetic peptide as well asto modulate the affinity of drug peptide compounds for their targets. Alimitation of the phage and bacterial display systems resides in theinability of these systems to incorporate amino acid analogs intopeptide libraries in vivo. In vivo, amino acid analogs disrupt thecellular machinery used to incorporate natural amino acids intoessential proteins as well as the growing peptide chain of interest.Phage and bacterial display both rely on the protein synthesis machineryof the bacterial cell to synthesize proteins essential for viability,synthesize the peptide library, and amplify or propagate the phage orbacterial pool harboring the peptide of interest. Technically cumbersomeprotocols can be time consuming when attempting the in vitro translationmethods frequently used to incorporate amino acid analogs into a peptidesequence.

The method of propagating the phage or bacterial pool requiresexpression of the peptide of interest. Peptides that are toxic to thebacterial cell and therefore lethal cannot be screened for in phage orbacterial display systems. This eliminates a potentially large segmentof peptides that otherwise would be of interest.

Phage and bacterial display also rely upon cumbersome and time consumingtechniques in order to keep conditions optimal for cell growth and cellviability. Bacterial cells are relatively large and care must be takenwhile screening for target interacting peptides. Affinity chromatographyis a common method used to separate non-binding peptides from bindingpeptides and care must be taken to prevent plugging and the non-specificretention of bacteria in the column. Candidate peptide displaying phageare generally amplified or propagated and therefore require the use ofthe cellular transcriptional, translational, and replication machineryof bacteria to synthesize the packaging proteins of the phage as well asthe peptide of interest. Infecting bacterial cells, harvesting thephage, and re-infecting several rounds is very time consuming. Thebacterial cell display system also requires optimal growth conditions toensure safe passage of the plasmid encoded peptide from generation togeneration and for subsequent re-screening.

Oligonucleotide-mediated mutagenesis has been utilized to furthercharacterize selected peptides. Generally, oligonucleotide-mediatedmutagenesis is used to introduce very specific mutations into the geneof interest. Although the selection of specific mutations to beintroduced into the gene is usually based on published reportsdescribing the effects of the mutations on the activity or function ofother homologous proteins, it is still difficult to predict the affectof the mutation or substitution.

It is often advantageous to increase the spontaneous mutation frequencyof the peptide library in vivo. Increasing the diversity of a populationof peptides displayed on a bacterial surface has proven to be a veryuseful tool for identifying those with a particular effect. Spontaneousmutations maintain evolutionary pressure on the peptide library andmaximize the screening of unique sequences.

A display system that is amenable to the uncomplicated nature of cloningand amplification of DNA sequences using the genetics of bacteria, forexample E. coli, to increase the variability and size of the peptideswithin the library is desirable. There is a need to generate novelpeptide libraries in a system that will allow the in vivo incorporationof amino acids analogs into the oligonucleotide sequence such that itsgenetic and biochemical characteristics are altered. There is a need forgenerating peptides that may otherwise be eliminated by virtue of theirtoxicity in phage or bacterial display systems. There is also a need tomanipulate the oligonucleotide in vivo and yet alleviate the requirementto ensure optimal growth conditions for cell viability.

It is therefore an object of this invention to provide an effective andrapid method for the systematic preparation of novel peptide substrateshaving altered functional and binding activity and to address theshortcomings inherent in the phage and bacterial display methodscurrently practiced in the art.

BRIEF SUMMARY OF THE INVENTION

Methods for selecting oligonucleotides and peptides of interest, andgenerating and screening large mini-cell display libraries for peptideswith desired functional and binding characteristics have been developed.These methods include selecting new and unique target interactingpeptides from minicell display libraries of random oligonucleotides thatare expressed as gene fusions to a protein such as the 17K antigen ofRickettsia rickettsii.

The plasmid or expression vector encoded oligonucleotide fusion or genefusion product is preferably localized to the minicell outer membraneforming what is referred to as a “display minicell”. Briefly, the methodconsists of first constructing a library wherein the library consists ofa replicable expression vector includes an inducible transcriptionalregulatory element operably linked to a gene fusion, where the genefusion includes:

(i) a first gene encoding at least a portion of a bacterial outermembrane protein; and

(ii) a second gene or oligonucleotide encoding a potential “substrate”peptide interacting with a target molecule.

The 3′ end of the first gene is linked to the 5′ end of the second geneor oligonucleotide, thereby forming a chimeric gene. The chimeric geneencodes a chimeric protein. The linkage between the first and secondgene may be direct, or indirect via a linker molecule oroligonucleotide. The second gene or oligonucleotide is obtained from alibrary of random oligonucleotides constructed by degenerate polymerasechain reaction (PCR), a method well known within the art, or otheramplification method.

In certain embodiments, it is desirable that the first gene encodes anouter membrane protein, or portion thereof, amenable to fusing largeoligonucleotides encoding proteins greater than 302 amino acids inlength. The 17K antigen of Rickettsia rickettsii is preferred. In oneembodiment the expression of the fusion protein is regulated by aninducible DNA regulatory element, for example, a lac promoter, tacpromoter (a hybrid trp-lac promoter that is regulated by the lacrepressor), trp promoter, or lacUV5 promoter. Other suitable microbialpromoters may be used as well. By using an inducible promoter, theoligonucleotide fusion will remain quiescent until the addition of theinducer. This allows control of the timing of production of the geneproduct.

The method further includes mutating the expression vector at one ormore selected positions within the second gene, thereby forming a familyof related substrate peptides encoded by the second gene. Next, suitablehost minicell strains are transformed with the expression vector DNApreparation. The method also provides for the induction of replicationof the acquired plasmid DNA and the controlled expression of thecorresponding peptide within the minicell.

Optionally, the method consists of transforming suitable host minicellstrains exhibiting a mutator phenotype and subsequent induction of theminicells to replicate acquired plasmid DNA. The method further includesgenerating a bacterial minicell strain exhibiting mutator phenotype.Mutations in genes responsible for DNA repair typically have a mutatorphenotype. For example, mutations in the genes responsible for themethyl-directed mismatch repair of DNA, designated mutS, mutL, and mutH,increase the spontaneous mutation frequency about 1000-fold.Incorporating one, two, or all three of these mutations into the parentbacterial cell results in the in vivo diversification of the peptidedisplay library within the anucleate minicell population.

The minicells are subsequently induced to express the library ofpeptides on their outer surface. The pre-selected target molecules arethen contacted with the display minicells and the peptide library isscreened for binding activity by methods well established within theart.

The pre-selected target molecule can be a protein, peptide,carbohydrate, sugar, nucleic acid, metal, or non-protein organicmolecule, such as a drug, vitamin or co-factor, neuromediator, cellreceptor or cell receptor complex, steroid, peptide mimicking a naturalacceptor binding site to a pre-selected molecule or an analog thereof,or an individual protein of a receptor complex.

In another embodiment, functional screening assays are incorporated toestablish biochemical activity relating to, for example, inhibitory,stimulatory, or responsive processes associated with the peptide ofinterest.

Those minicells that bind to the target molecule are separated fromthose that do not. Optionally, the peptides displayed on the minicellsmay be labeled with molecules or compounds such as radioactive isotopes,rhodamine, or FITC before, during, or after expression of the displaylibrary. This serves to facilitate subsequent identification of thebound peptide of interest. For example, antibodies available to thetarget molecule may be used to immunoprecipitate the interactingcomplex. If the interacting peptide is radiolabeled, the complex can beeasily distinguished and visualized by autoradiography, a method wellestablished within the art. Optionally, the minicells may besupplemented exogenously with amino acid analogs to be incorporated intothe peptide being synthesized in vivo.

The bound minicell library members that have been separated from theunbound members now represent an enriched library. The expressionvectors that contain the oligonucleotides of this enriched library canbe isolated, mutagenized and displayed again to screen for alteredspecificity of the fusion protein towards the target. Alternatively, theenriched library may be tested again, under more stringent conditions,for binding ability, those that bind are separated from those that donot and the library is further enriched.

This method may be repeated one or more times with either the minicellsthat bound to the target molecule or those that did not.

The bound minicells can be easily eluted from the target molecule andthe peptide encoding expression vectors isolated to extract information.The DNA sequence of the peptide, DNA base composition, the molecularweight, and/or whether any secondary structures exist within thesequence can then be determined.

Optionally, the method comprises liberating the peptide of interest fromthe display protein, to which it is genetically fused, for subsequentamino acid analysis. Amino acid analysis of the peptide library iscarried through by methods well known within the art using automatedanalyzers. One can also determine the amino acid composition, the aminoacid sequence, the isoelectric point, and molecular weight of thepeptide.

These peptides can then be further screened for desired activities.Further rational manipulation can also be performed to delete, add, orsubstitute specific amino acids or to label the peptide or to immobilizethe peptide for use in diagnostic screening assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the strategy for displaying random librarypeptides on the surface of minicells.

DETAILED DESCRIPTION OF THE INVENTION

I. Minicell construction and composition.

Minicells offer an alternative method for packaging library DNA anddisplaying peptides. As used herein, the terms “peptide” and “protein”are used interchangeably unless otherwise noted. Minicells are small,anucleate cells resulting from aberrant cell divisions at the polar endsof bacteria. However, the minicells are large enough to harbor severalplasmids and have been extensively used to analyze cloned proteinexpression since they lack bacterial chromosomal DNA, but contain all ofthe necessary machinery for coupled transcription and translation, andprotein modification. Many mutant bacterial strains, representing grampositive and gram negative strains, are capable of producing minicellsthroughout their respective cell cycles. Examples include E. coli, S.typhimurium, S. anatum, S. enteritidis, S. pullorum, S. senftenberg, S.worthington, B. subtilis, V. cholera, E. amylovora, and H. influenzae.

A) Min mutations.

Bacterial cells have been provided with an elegant system to controlcell division. The min genes provide bacteria with the ability tocontrol where, anatomically, cell division will take place. Whenbacteria normally divide, min proteins (MinC, MinD, and MinE) accumulateat the two polar ends of each cell. The min proteins prevent the celldivision apparatus from accumulating at the ends of each cell and can bethought of as polar cell division inhibitors. MinE provides thetopological specificity required for correct localization of the MinCand MinD proteins to the cell poles. See de Boer et al., Proc. Nat.Acad. Sci., (87) 1129–1133 (1990) or de Boer et al., Cell, (56) 641–649(1989). With the polar ends of each cell blocked from division apparatusassembly, the proteins required for division accumulate in the middle ofthe cell (midcell). Cells lacking any of the minC or minD genes, oroverexpressing the MinE protein, aberrantly divide at the polar endswith increased frequency, forming chromosomal DNA deficient minicells.The formed minicells, while unable to divide, are able to incorporatenucleotides into replicating plasmid DNA and synthesize protein encodedby the sequences of the plasmid. Any bacterial strain capable of formingminicells can be used as a bacterial host for the expression of thedisplay peptide.

The principle components of the bacterial minicell strain includemutation(s) in gene(s) that confer the minicell phenotype. The mutationsare preferably in a genetically clean genomic background (only thosemutations conferring desired phenotype(s) are present in an otherwisewild-type background).

B) Mutator mutations.

In another embodiment, an in vivo method for further randomizinglibraries of diverse oligonucleotides and the peptides encoded by themis used. A mutation in the mutS gene that renders the encoded proteinnon-functional also renders the cells harboring the mutation incapableof correcting mistakes made during DNA synthesis/replication. A mutSstrain will confer a mutator phenotype.

The peptide libraries can be further diversified in vivo utilizingmutations in one of the min genes, for example minC, and transducing themutant gene into a mutS cell line. The newly created cell line (MsMc)harbors mutations in both mutS and minC genes. Using techniques such ascalcium chloride transformation or electroporation, the oligonucleotideharboring plasmid can be introduced into the new cell line. Thetransformed cell line may be induced to replicate plasmid DNA, byexogenously adding nucleotides, and in doing so the replicationmachinery of the minicell will incorporate or substitute a mis-basepaired nucleotide at a rate of approximately one per one thousand basescopied or replicated. Therefore, 5×10⁸ bacteria will generate 10⁵ newsequences every generation. The plasmids can then be transferred to anon-mutator minicell strain for further display.

C) Amino Acid Analogue Incorporation

Providing display minicells with amino acid analogues to be incorporatedinto the peptide of interest can be used to further diversify thelibrary. In order for amino acid analogues to be incorporated into thepeptide, the tRNA molecules involved in synthesizing the peptide frommRNA must be modified. tRNA molecules serve to chemically linkthemselves to a particular amino acid and then present the amino acid,corresponding to the correct sequence in the mRNA, for incorporationinto the peptide chain. Twenty aminoacyl-tRNA synthetase enzymes, eachcorresponding to one of the twenty naturally occurring L-amino acids,add amino acids to accepting tRNA molecules. Mutations may beincorporated into any one, several, or all, of the genes encoding theaminoacyl-tRNA synthetases of the MsMc strain that will allow them torecognize and transfer analogues of amino acids to corresponding tRNAmolecules. The resultant tRNA molecules then have the ability toincorporate an amino acid analogue into the growing peptide chain.Alternatively, the tRNAs may be genetically constructed to be recognizedonly by the synthetases that will aminoacylate with the amino acidanalogue and be directed to recognize nonsense codons (suppressor tRNAs)or four base codons. See Magliery et al., J. Mol. Biol., March 2001,307(3): 755–769. Such a combination will provide for specific in vivoincorporation of an amino acid analogue (Wang et al., Science, April2001, 292:498–500; Liu and Schultz, Proc. Natl. Acad. Sci. USA, April1999, 96:4780–4785). Amino acid analogues such as any hydroxyamino acidor derivative thereof, ornithine, azitryptophane, or D-amino acids canbe supplied exogenously to the cells to be incorporated into the peptidechain. Alternatively, any minicell strain may harbor mutations in genesencoding the tRNA molecules.

II. Plasmid Construction.

Generally, the plasmid used is able to serve as a cloning vector that issuitable for replicating in the desired host strain. The origin ofreplication and control sequences are compatible with the host minicellto be used for display. For example, the plasmids pUC19 or pBR322, orderivatives thereof, may be used if E. coli is the strain (parent) fromwhich the minicells are derived. The plasmids preferably include aselectable marker gene or genes that is able to be selected in theparent host. A selectable marker gene includes any gene that confers aphenotype on the parent cells to be selectively grown. Examples ofselectable marker genes include, but are not limited to, thetetratcycline gene, the kanamycin gene, the ampicillin gene, and thegentamycin gene. It is preferred that the plasmid contain an inducibleregulatory element for the controlled expression of sequences ofinterest. The plasmid should also be amenable to cloning DNAoligonucleotides, for example, ranging in length from 9 base pairs to3000 base pairs, and be able to serve as a template for expression ofoligonucleotide fusion proteins. The plasmid should also exist inmultiple copies within the host cell typically ranging from 2 to 100copies per cell.

III. Peptide Fusion Construction

A peptide capable of binding a target molecule is obtained from a randomminicell library wherein the minicells express fusion proteins includingat least one random peptide sequence joined to a protein exposed on theouter surface of the minicell. The fusion may be direct, or indirect vialinker sequences. Indirect linkage can be represented by the directchemical coupling between the outer membrane protein and the substratepeptide. For example, one of ordinary skill in the art will realize theplethora of nucleic acid linkers commercially available and,alternatively, available by de novo construction (it is not necessarythat such a linker represent a sequence of amino acids that is normallyfound on the surface of a cell).

The fusion (chimeric) protein to be displayed on the surface of theminicell is generally cloned into the plasmid expression vector fromwhich the chimeric gene encoding the chimeric protein will be expressed.

A) First gene.

The peptide to be used to direct the second gene product to the minicellsurface is usually selected because it encodes a signal amino acidsequence capable of mediating correct localization of the fusion, orchimeric, protein to the outer surface of the minicell. Signal sequencesinclude, for example, ompA signal sequence, ompT signal sequence, ompFsignal sequence, ompC signal sequence, beta lactamase, the traA signalsequence, the phoA signal sequence, and the 17K antigen signal sequenceof Rickettsia rickettsii. Furthermore, peptides harboring signalsequences that are not normally associated with the outer membrane maybe modified with lipid modification consensus sequences to ensureattachment to the outer membrane.

A preferred peptide consists of the first 71 amino acids (213nucleotides) of the 17K antigen open reading frame (ORF) of R.rickettsii, contains the signal sequence as well as a lipid modificationsite. The 213 base-paired oligonucleotide (SEQ ID NO:6) may be assembledby annealing different regions of primers corresponding to overlappingregions of the first 213 nucleotides, forming a concatamer of DNA.Single stranded portions of the concatamer are subsequently converted todouble strands by purified enzymes known in the art. The resultingdouble stranded DNA can then be cloned into the appropriate plasmidexpression vector to generate a genetic fusion to an inducibleregulatory promoter element. Such a process may be used to clone anynucleic acid sequence encoding a peptide that localizes to the outermembrane of the minicell. The membrane protein encoding sequence, forexample, the 213 nucleotide fragment encoding the first 71 amino acidsof the 17K antigen ORF, is preferably positioned downstream of thepromoter and upstream of the oligonucleotide (second gene) encoding thepeptide of interest. Any termination sequence that is recognized by theexpression machinery of the minicell may be used to terminatetranscription. It is well known in the art that bacterial DNA sequences,and plasmid DNA sequences, rely upon one of two basic types oftranscription termination, factor independent and factor dependent(based upon whether the RNA polymerase requires more than just thesequence to terminate). Such termination sites are applicable to thedisclosed constructs.

B) Peptide to be targeted (second gene).

The oligonucleotide library encoding the randomized peptide to betargeted can be synthesized in vitro using PCR or other amplificationmethods that are well established within the art. In the preferredembodiment, the library includes at least about 10¹⁰ oligonucleotideswhich encode the peptides. Generally, the oligonucleotide librariesinclude a unique or variable sequence region which confers diversity tothe library. Diversification of the library is typically achieved byaltering the coding sequence which specifies the sequence of the peptidesuch that a number of possible amino acids can be incorporated atcertain positions. At the core of creating the library lies theconstruction of degenerate primers. Degenerate primers can beconstructed using available automated polynucleotide synthesizers, suchas one of the Nucleic Acid Synthesis Instrument Systems (AppliedBiosystems).

Primer sequence may be made up of a specific series of nucleotides ortheir equivalent IUB codes (for example, R {A,G}, W {A,T}, K {G,T}, M{A,C}, S {G,C}, V {A,G,C}, D {A,G,T}, H {A,C,T}, B {G,C,T} and N{A,G,C,T}). Many systems have been programmed to recognize IUB ambiguitycodes such that an input sequence of DDDD would correspond to a fourbase primer sequence with each position having an equal probability ofan A, G, or T incorporated. Once constructed, the randomized primerswill contain regions of complementarity, within their sequence, to otherprimers. The complementary primers are annealed forming concatamers ofnucleotide sequence whose single stranded gaps are filled in withnucleotides and polymerase to form randomized double strandedoligonucleotides. The double stranded oligonucleotides can then becloned into the expression plasmid downstream of the inducible promoterand preferred 17K antigen to form the chimeric gene fusion.

Alternatively, oligonucleotides may be mutagenized in vitro using wellknown methods in the art. In vitro mutagenesis of oligonucleotides,oligonucleotides encoded within a plasmid, or gene fusions harboring theoligonucleotide in a vector or plasmid, may be site directed or random.The mutagenized plasmid can then be used to transform the minicells orminicell strain for subsequent induction of expression and screening forbinding activity of the encoded peptide.

In another preferred embodiment, the bacterial minicell strain istransformed with the newly constructed plasmid. Transformation methodsinclude, for example, phage transfection (e.g. P1, lambda, or M13),electroporation, and transformation. It is preferred that the parentminicell strain be transformed, selected via a selectable marker on theplasmid, and minicells isolated from the parent strain harboring theplasmid. Alternatively, the isolated minicells from a parent strain maybe directly transformed.

IV. Minicell Isolation and Display Induction

A) Minicell Purification

In a preferred embodiment, cells harboring one or more min mutationsthat have undergone the desired asymmetric cell division (polar celldivision) are separated from those that have not. Minicells areseparated from whole bacterial cells based on their difference in sizeand density. Density gradient centrifugation is used to separate andisolate minicells from the population of “whole” cells present in theculture. Isolated minicells remain stable and active for 48 hours atroom temperature or up to 6 weeks at −70° C. Room temperature stabilityeliminates the need for time consuming protocols that are required tokeep whole cells and phage growing in optimal conditions throughoutdisplay methods known in the prior art. Minicells are physiologicallynot capable of cell division.

B) Replication of Plasmid DNA.

In a preferred embodiment, the transformed minicells are induced toreplicate the plasmid DNA by exogenously adding nucleotides required forincorporation into the growing DNA strand. Replication of the plasmidDNA increases the plasmid copy number within the cell. If thetransformed cells harbor a mutator phenotype the diversity of thepeptides to be displayed on the outer surface will increase. The mutatorphenotype exhibits its effects at the nucleotide level of DNA synthesiscompared to another diversification technique, the incorporation ofamino acid analogues at the level of peptide synthesis.

C) Induction of Chimeric Protein Expression.

The expression of the peptide to be displayed is under the control of aninducible promoter. Many inducible promoters are available in the artfor controlling gene expression and can be used herein. Induciblepromoters are ideal for the expression of peptides that would otherwisebe toxic to normally dividing bacterial cells. The toxic peptides thatwould normally kill a dividing cell cannot exert their lethal effectswithin the minicell. Minicells are not growing, or dividing, and lackchromosomal DNA. Minicells are isolated and subsequently induced forexpression of the chimeric peptide. The induction is usually carried outby exogenously supplying the minicells with amino acids and an inducerthat will activate protein expression. For example, addition of theinducer isopropylthiogalactoside (IPTG) will relieve repression of genesunder the control of lac, tac, or lacUV5 promoters. These promoters arenegatively regulated by the lacI repressor protein when the addition ofIPTG is omitted. The exogenously added amino acids provide the subunitsrequired for growth of the peptide chain. Optionally, amino acidanalogues may be added simultaneously or in place of the L-amino acidsto further diversify the peptide library to be displayed.

V. Interactions Between Peptides to be Screened and Target or BindingMolecules (“binding partners”).

The displayed peptide and interacting molecule or target are screenedfor an interaction. The interaction requires binding between the peptideencoded by the second gene and the target molecule. The peptide may be asubstrate, cofactor, ligand, or effector. The target molecule may be apeptide or protein, nucleic acid molecule, carbohydrate or sugar,vitamin cofactor, metal, or synthetic drug. The target molecule may be asubstrate for an enzyme, a cofactor that forms part of a functionalcomplex, an enzyme which acts on the peptide encoded by the second gene,or a ligand or receptor interacting with the peptide encoded by thesecond gene. Examples of such target molecules include peptideinteracting pairs which include antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate, IgG-proteinA. The target molecule that interacts with the displayed peptide mayalternatively be part of a library of random peptides. Preferably, thestrength of the binding reaction is sufficient to allow the interactingpair to be isolated based on the physical reaction between the targetand the random peptide. A pre-selected “target” molecule can be a drug,vitamin neuromediator, cell receptor or cell receptor complex, steroidhormone, metal, carbohydrate, inorganic or organic compound, peptidemimicking a natural acceptor binding site to a pre-selected molecule oran analog thereof, or an individual protein of a receptor complex.

VI. Separating Bound Minicells from Unbound Minicells.

In methods analogous to affinity chromatography, the pre-selected targetmolecule, or library of random peptides, (binding partner(s)) may beimmobilized by attaching it to a suitable solid support matrix such asagarose beads, acrylamide beads, cellulose, neutral and ionic carriers,or various acrylic polymers. Methods used to attach the pre-selectedmolecule or library to a particular matrix are well established withinthe art and described, for example, in Methods in Enzymology, 44 (1976).After attachment of the molecule or peptides to the matrix, the isolateddisplay minicells are incubated with the matrix, allowing contact to bemade between the minicell and the binding partner. Unbound cells arewashed away and the minicell bound to the pre-selected target may beeluted by a variety of methods including adjusting pH conditions, ionicconditions or by competing with excess free antigen. Elution conditionsthat may otherwise be detrimental to bacterial growth and vitality maybe incorporated when eluting display minicells. Growth and vitality arenot at issue with minicells. The relatively large size of bacterialcells may also preclude one from using affinity chromatography becauseof plugging of columns used in the technique. The smaller size ofminicells is amenable to affinity chromatography.

VII. Peptide Analysis.

The displayed peptide library may be analyzed to determine the diversityand/or composition of the amino acids incorporated. Minicells may besubjected to enzymes or acids known to specifically cleave betweencertain peptide residues to release the peptide of interest from thedisplay chaperone protein (for example, formic acid cleaves peptidebonds between proline and glycine residues). The peptides are thenhydrolyzed and analyzed for amino acid content using automated aminoacid analyzers.

The peptides may also be analyzed for precise amino acid sequence. Forexample, the classic method of Edman degradation, in which theN-terminus of the peptide becomes modified, cleaved, and analyzed, thusshortening the peptide by one amino acid, is one way of extractinginformation at the amino acid level. Mass spectrometry is a moresophisticated technique and amenable to analyzing peptides that haveincorporated amino acid analogues. Mass spectrometry utilizes helium gasto randomly cleave the peptide and subsequent analysis of the mass ofthe fragments generated are compared to elucidate the sequence. Thepeptide sequence can then be used to determine and/or designoligonucleotides encoding the peptides.

VIII. Oligonucleotide (second gene) Analysis.

Because minicells are amenable to the uncomplicated nature of bacterialgenetics, it is relatively easy to isolate the plasmid expression vectorfrom the minicell by methods known to those skilled in the art and, ifdesired, to further propagate the plasmid in a suitable host.Alternatively, the second gene sequence contained within the isolatedexpression vector may be directly amplified by PCR and sequenced, usingprimers to known sequence within the 17K antigen (first gene) and/or theparent expression vector.

Once isolated, the plasmid expression vector may be mutagenized in vitroto study the effects of specific mutations in the genes encoding thepeptide of interest. Such effects can be assayed genetically, orbiochemically, as discussed below. Site directed and random mutagenesisof plasmids and vectors are well established in the art.

In another embodiment, the method uses a vector suitable for fusingoligonucleotide libraries with the display “chaperone” DNA. Thepreferred chaperone DNA encodes the 17K antigen of Richettsiarickettsii.

IX. Screening Peptides for Activity.

The peptides are preferably isolated or identified based on binding. Thepeptides may also, or alternatively, be screened for bioactivity. Abioactivity can be any biological effect or function that a peptide orprotein may have. For example, bioactivities include specific binding tobiomolecules (for example, receptor ligands), hormonal activity,cytokine activity, and inhibition of biological activity or interactionsof other biomolecules (for example, agonists and antagonists of receptorbinding), enzymatic activity, anti-cancer activity (anti-proliferation,cytotoxicity, anti-metastasis), immunomodulation (immunosuppressiveactivity, immunostimulatory activity), anti-infective activity,antibiotic activity, antiviral activity, anti-parasitic, anti-fungalactivity, and trophic activity. Bioactivity can be measured and detectedusing appropriate techniques and assays known in the art. Antibodyreactivity and T cell activation can be considered bioactivities.Bioactivity can also be assessed in vivo where appropriate. This can bethe most accurate assessment of the presence of a useful level of thebioactivity of interest. Enzymatic activity can be measured and detectedusing appropriate techniques and assays known in the art.

As demonstrated by Example 9, several (second gene) peptides that bindto receptors that are found on the cell surface and are required fortumor metastasis have been identified using this system. These potentialmetastasis blocking peptides have been further evaluated for effecting aparticular response on the receptor that can be assayed biochemically.Peptides have been shown to influence the autophosphorylation ofreceptors in vitro, by assaying the amount of radiolabeled phosphateretained by the receptor before and after interaction with the peptide.This can be shown using standard techniques within the field ofmolecular biology. By influencing the phosphorylation of cell surfacereceptors the isolated peptides can directly influence the activity ofthe cellular processes these receptors control. Methods are wellestablished in the art that allow post translational, or peptidemodification, of the isolated peptides in vitro. Such modificationsinclude, but are not limited to, acylation, methylation,phosphorylation, sulfation, prenylation, glycosylation, carboxylation,ubiquitination, amidation, oxidation, hydroxylation, adding aseleno-group to amino acid side chains (for example, selenocysteine),and fluorescent labeling.

Further in vitro analyses are used to study the effects of the peptideson cell viability. Peptides that either interrupt, stimulate, ordecrease vital cellular processes may be used to infect cells, such astumor cells, in culture. Once infected, cell growth and viability isanalyzed by methods known in the art.

Many cells may undergo programmed cell death which is a geneticallymediated form of self destruction. This phenomenon is commonly referredto in the art as apoptosis. Frequently, apoptotic cells may berecognized by changes in their biochemical, morphological and molecularfeatures. Morphological changes include, but are not limited to, cellshape change, cell shrinkage, cell detachment, apoptotic bodies, nuclearfragmentation, nuclear envelope changes and loss of cell surfacestructures. Biochemical changes may include proteolysis, protein crosslinking, DNA denaturation, cell dehydration, intranucleosomal cleavageand a rise in free calcium ions. Such characteristics are easilyidentifiable by methods well established in the art. Peptides isolatedby the disclosed mini-cell display method are tested for their effectson such physiological and biochemical processes.

When cells are no longer viable, i.e. they are dead, their membranesbecome permeabilized and this permeabilization will manifest itself as achange in the scattering of light. This scattering of light can beattributed to the change in the refractive index of the cell'scytoplasm. The use of DNA staining dyes that are able to pass through apermeabilized membrane, will aid in the identification of dead, live,and apoptotic cells. Flow cytometry and/or fluorescent activated cellsorting (FACS analysis) may be incorporated into protocols utilizingfluorescent dyes to separate the cells of interest. Flow cytometry cansort, or physically separate, particles of interest from a sample.Therefore, FACS analysis (which is a type of flow cytometry), may bedefined as the physical separation of a cell or particle of interestfrom a heterogeneous population.

One may distinguish between dead, live, and apoptotic cells because eachdiffer, for example, in their permeability to DNA dyes. Two widely usedDNA dyes, Hoechst 33342 and propidium iodide (PI), are able toinfiltrate dead cells. Live cells do not retain either dye, whileapoptotic cells are able to retain Hoechst but not PI. Fluorescentmicroscopic observation will allow one to visually separate dead cellsfrom live cells from cells undergoing apoptosis. Fluorescence emissionfrom these different cells will also allow their separation via flowcytometry and/or FACS analyis. Typical stains used in these assays willinclude, propidium iodide, Hoechst 33342, 7AAD and TO-PRO-3.

Stages of membrane change during apoptosis may be analyzed as well.Among these changes is the translocation of phosphatidylserine (PS) fromthe inner part of the cell membrane to the outside during the early tointermediate stages of apoptosis. Using FITC labeled Annexin V, one maybe able to detect PS. Annexin V is a Ca⁺⁺ dependent phospholipid-bindingprotein. Again, dead cells will not bind Annexin V. Live cells are alsonegative for Annexin Binding. Apoptotic cells bind Annexin. One maycombine this method of analyzing PS with the aforementioned method ofusing PI to stain DNA, thereby obtaining different profiles of live,dead, and/or apoptotic cells.

As mentioned above, a characteristic of apoptosis is the degradation ofDNA. This degradation is usually carried out by activated Ca/Mgdependent endonucleases. Terminal deoxynucleotidyl transferase (TdT)will add biotinylated, BrdU or digoxygenin-labeled nucleotides to DNAstrand breaks. Subsequent binding of the exogenously added streptavidinby the biotin, or a fluorochrome labeled anti-digoxygenin antibody maybe used to then detect DNA degradation. This method allows one tocorrelate apoptosis with cell cycle status.

Another DNA binding dye that may be incorporated is the laser dyestyryl-751 (LDS-751). Again, one may take advantage of the ability ofapoptotic cells to exhibit different staining patterns than that of liveor dead cells.

Laser capture micro-dissection (LCM) is a relatively new technology usedfor the procurement of pure cells from various tissues. Isolated tissuesmay be used to identify what effects a peptide may have on cells thathave either internalized the peptide or have bound the peptide to anouter surface receptor. After transfer film is applied to the surface ofa particular tissue section, one may activate a pulsed laser beam that,in turn, activates the film immediately above the cell(s) of interest(morphological changes are easily identified and cells may be selectedon this basis). The film melts and fuses the underlying cells. The filmcan then be removed and the remaining cells, not contained within thefilm, are left behind. Once the cells are isolated, DNA, RNA or proteinfrom the cells may then be purified. The isolation of the cells via LCMdoes not damage the cells because the laser energy is absorbed by thefilm. This particular technology may be useful in combination with anyof the previously mentioned methods of detecting proteins usingfluorescent molecules.

In vivo analyses using animal models are used to determine the effectsof the peptide within an intact system. For example, in the field ofimmunology, peptides can be administered to an animal and its peripheralblood monocytes are used in the generation of antibodies directedagainst the peptide.

In the case of viral proteins—for use with, for example, viral vectors,therapeutic viruses, and viral capsid delivery compositions—desiredcharacteristics to be retained can include the ability to assemble intoa viral particle or capsid and the ability to infect or enter cells.Such characteristics are useful where the delivery properties of theviral proteins are of interest.

One application of the disclosed method is in the identification anddevelopment of peptides, and the oligonucleotides encoding thosepeptides, for use in subsequent gene replacement and/or gene enhancementtherapy. For example, identifying anti-tumor peptides that specificallytarget the receptors involved in the metastatic spread of tumors. Targetinteracting peptides have been successfully isolated and identifiedusing the minicell technology.

Invasion complexes have been shown to play a prominent role in cellularactivities such as regulating actin and microfilament rearrangementswithin the target cell, and therefore playing critical role in pseudopodformation, as well as shutting down DNA synthesis and replication. Theinhibition of DNA replication would then have a direct impact onapoptosis.

Invasion complexes also regulate normal and abnormal cell proliferation(for example, cancer cell metastasis and replication). Chemotaxis,migration and other modes of cellular recruitment and motility are alsoregulated by cellular interactions with invasion complexes. For example,egg fertilization may be inhibited or enhanced by such interactions.

Using the methods and materials described herein, one of skill in theart can isolate invasion complexes using proteins to which thecomplexes, normally or abnormally, bind as targets. For example, MCP-1,RAMF (a receptor for hyaluronic acid), glycosaminoglycans (GAG), andosteopontin (to isolate CD44 splice variants) may used to isolate wholeor partial complexes. The isolated complexes can be used to screen forinhibitors of activity, using the minicell library technology describedherein. Alternatively, peptides that bind to and either inhibit orenhance invasion complex activity may be identified using the disclosedmini-cell display technology.

The present invention will be further described below by way of thefollowing non-limiting Examples and appended figures.

EXAMPLE 1 Construction of a 17K Antigen Fusion Plasmid for MinicellDisplay

A system was constructed to allow the controlled expression ofoligonucleotide libraries genetically fused to the 17K antigen ofRickettsia rickettsii. The 17K antigen of R. rickettsii, when clonedinto E. coli is displayed to the outer membrane. The N-terminalfragment, containing the lipid modification site, was assembled from thefollowing primers and cloned into pZHA1.3, a plasmid derived from pUC19,by inserting the tac promoter upstream of the unique HindIII site.

Primers were dissolved in 10 mM Tris, pH 8.5, to a concentration of 100nmol/μl. 10 μl of each was then mixed, heated to 80° C. for 5 min,cooled to 25° C. (ramp time 1 hour), and incubated at 25° C. for 1 hour.The annealed oligonucleotides were filled in with Klenow, and purifiedusing a QIAquick PCR purification kit (Qiagen), before restrictiondigestion. The resulting double stranded DNA was cut with XbaI/BamHI andligated overnight at 14° C. into the XbaI/BamHI of pZHA1.3 to formpZHA2.0.

The bold lower case bases of Primer 1 represent the Xba1 recognitionsite. The bold lower case bases of Primer 4 represent the BamHIrecognition site. The bold upper case bases represent complementarybases used to generate double stranded sequences upon annealing. Primer1 contains bases complementary only to Primer 2. Primer 2 contains basescomplementary to Primer 1 and Primer 3. Primer 3 contains basescomplementary to Primer 2 and Primer 4. Primer 4 contains basescomplementary only to Primer 3.

Primer 1 (SEQ ID NO:1) tctagaATGAAACTTTTATCTAAAATTATGATTATAGCTCTTGCAACTTCTATGTTAGCCGCC Primer 2 (SEQ ID NO:2)TCGGCGGACATTGCCAGGCCCGCCATACTTATTTGTTCCATGTC CTTGTGAAGAACCGCCACGACCGPrimer 3 (SEQ ID NO:3) GGCGGTGCTGGCGGCGCATTACTTGGTTCTCAATTCGGTAAGGGCAAAG Primer 4 (SEQ ID NO:4)CCCGTTTCCTGTCGAACAACCTCATCCACATCCAGGTAATGAACCTCGTCAAGAACCACCTGTTTAGCCggatccThe resulting plasmid, PZHA2.0 expresses the first 71 amino acids (SEQID NO:5) of the 17K antigen of R. Rickettsii (DNA encoding the first 71amino acids is shown in SEQ ID NO:6), under the control of an IPTGinducible promoter (tac promoter). This vector was used for constructionof the display library.

EXAMPLE 2 Construction of the Library

The primers were synthesized on an Applied Biosystems synthesizer(Forest City, Calif.).

1 mM of each primer was separately incubated in 100 μl of 10 mM Tris-HClbuffer, pH 8.0, containing 5 mM MgCl₂, 0.5 mM dNTPs and 5 U of Tacpolymerase. Reactions were heated to 80° C. for 5 min, cooled to 25° C.(ramp time 1 h), and incubated at 40° C. for 15 min. This procedure wascycled 5 times. After the fifth cycle, 10 μl of each reaction mix wasmixed pair-wise with 10 μl of samples from the other reaction asillustrated below. The total volume of each reaction was adjusted to 100μl with Tris buffer, pH 8.0 containing 5 mM MgCl₂, 0.5 mM dNTP, and 5 Utac polymerase and cycled as described above. After the fifth cyclefresh 5 μl of 100 mM dNTP mix was added to each tube and the chainingreaction continued for another 10 cycles.

The reaction mixes from all 42 tubes (the original 6 primers and the 36pair-wise tubes) were mixed (see table below) and double-strandedoligonuleotides, generated from the annealing of complementary regions,were purified as described above. The purified double strandedoligonucleotides were then incubated with 10 ug of pZHA2.0, previouslydigested with SmaI and tailed with dTTP and terminal transferase (togenerate a 5′ T overhang), and ligated overnight at 13° C.

Minicell E. coli strain DS410 was transformed with 5 μg of the resultingplasmid, pDIP1.0. The transformed bacteria were incubated over night in10 ml of LB broth containing 200 μg/ml ampicillin. This culturerepresented the display library.

EXAMPLE 3 Purification and Labeling of Minicells

Plasmid pDIP1.0 was transformed into E. coli DS410. Transformed cellswere grown in a 250-ml culture to stationary phase (the culture can begrown overnight but must have good aeration). Growing the cells in richmedium minimizes contamination of mini-cells by whole cells. The culturewas spun down at 8200×g (7500 rpm in a Sorvall GSA rotor) for 20 minutesat 4° C. The cell pellet was resuspended in 5 ml of the supernatant.Resuspension was very thorough to prevent loss of minicells in the cellpellet during the sucrose gradient step. Pellet was resuspendedcompletely by placing a magnetic stirring bar in the bottom of thecentrifuge tube and mixing vigorously for 10 minutes at 4° C. Thesuspension was carefully layered on a 30-ml sucrose gradient (10–30%).The gradient was centrifuged in a cellulose nitrate ultracentrifuge at4000×g for 20 minutes at 4° C. (e.g., in an SW27 rotor at 5500 rpm).After centrifugation, a thick, somewhat diffuse, white band of minicellswas visible near the middle of the tube. Minicells were collected (theband) from the side with a 20 cc syringe. Minicells were removed fromthe sucrose solution by centrifugation at 20,000×g for 10 minutes at 4°C. (13,000 rpm in the Sorvall SS-34). The minicell pellet wasresuspended in 1 ml of Methionine Assay Medium (Difco). The suspensionwas layered onto a 10-ml or a 30-ml sucrose gradient (10–30% gradient).The sucrose gradient was centrifuged at 4000×g for 20 minutes at 4° C.(e.g., in an SW40 rotor at 5700 rpm for a 10-ml gradient). The minicellband was removed as described above. Minicells were checked under themicroscope for contamination. If any whole cells are observed within themicroscope field, the minicells must be purified through another sucrosegradient. No contamination was observed and minicells were spun down asdescribed above. Pellets were resuspended in 1 ml of Methionine AssayMedium. The optical density was read at 600 nm. If the minicells are tobe used immediately, Assay Medium is added to give a concentration ofA₆₀₀ 2.0/ml. If not, the minicells are spun 1–2 minutes in a microfugeand the pellet resuspended in enough Assay Medium containing 30%glycerol to give O.D.₆₀₀=2. Store at −70° C. Minicells are generallyactive for at least 6 weeks.

EXAMPLE 4 Minicell Labeling and Induction of Expression

If the minicells to be labeled have been freshly prepared, they can beused directly (at a concentration of O.D.₆₀₀=2). Minicells that havebeen previously frozen, first need to have the glycerol removed. In thiscase, the minicells are pelleted by centrifugation in a microfuge for1–2 minutes. Minicell pellets are dissolved in enough Methionine AssayMedium to give O.D.₆₀₀=2. For each sample to be labeled, 250 μl wasplaced into a microfuge tube. 5–10 μCi of ³⁵S-methionine (alternatively,cold methionine may be used) was added to a volume of 1–2 μl, then 0.1mM IPTG was added to induce the library. Cells were incubated at 37° for90 minutes. Cells were chilled on ice and spun 1–1½ minutes in amicrofuge. Supernatant was removed and discarded in the radioactivewaste. Pellet was resuspended in 50–100 μl of 0.12M Tris, pH 7.1. Steps5 and 6 were repeated two more times for a total of three washes. Afterthe last wash, the pellet was resuspended in 20 μl 0.12 M Tris, pH 7.1.

EXAMPLE 5 Screening of Library for Bioactive Peptides

Several screening methods were utilized. Most of the methods follow asimilar protocol outlined below.

a. Immunoprecipitate the target receptor

b. Immobilize the receptor onto immuno-plates

c. Incubate the plate with freshly isolated minicells

d. Wash away unbound minicells

e. Elute minicells from the plate and transform the plasmids isolatedinto fresh minicell strain DS410 for second cycle of screening.“Positive” clones selected are then constructed into a secondarylibrary.

A tertiary library may be constructed from a third round of screeningand those peptides selected may be used in functional screening assaysto further isolate peptides of specific activity.

EXAMPLE 6 Amino Acid Analysis of Isolated Display Peptides

Minicells (2 A₆₀₀/ml) were resuspended in 1 ml 0.5 M formic acid (whichcleaves between proline and glycine and releases the library from thedisplay protein) then filtered through 1 KD cut off filtron NANOSEP™filter to isolate peptides of greater than 1000 dalton MW. Samples werehydrolyzed under vacuum in 6 N HCl at 104° C. for 18 hours. Thehydrolyzed samples were dried under vacuum and then reconstituted to 0.5ml in amino acid analysis buffer. The samples were analyzed for aminoacid content on a Beckman automatic amino acid analyzer using 0.2 Msodium citrate, pH 1.5 as the eluting buffer. The results (table 1,shown below) show that, as expected, amino acids were evenly distributedthroughout the sample (ser, thr, trp, and met are unstable under theseconditions and suffer extensive degradation).

TABLE 1 Amino acid analysis of display library. AA AA MWt nmol/mlres/1000 μg aa/ml MRW Calc Glu 147.13 10.399 61.8203 91.530005 0.420176Gln 146.15 0 0 0 0 Asp 133.1 11.64 69.1978 1.549284 0.519895 Asn 132.1 00 0 0 Hyp 131.3 0 0 0 0 Leu 131.17 9.565 56.8623 1.254641 0.433502 Tyr181.19 7.948 47.2492 1.440098 0.260774 Phe 165.19 8.832 52.5046 1.4589580.317845 His 155.16 12.714 75.5824 1.972704 0.487128 Lys 146.19 12.3273.2407 1.801061 0.500995 Trp 204.22 0 0 0 0 Arg 174.2 10.468 62.23021.823526 0.357237 HyLys 162.19 0.703 4.17925 0.11402 0.025767 Pro 115.139.285 55.1977 1.068982 0.47944 Thr 119.12 5.261 31.2752 0.62669 0.262557Ser 105.09 6.766 40.2221 0.711039 0.382746 Gly 75.07 10.7 63.60930.803249 0.84734 Ala 89.09 11.343 67.4326 1.010548 0.756902 Cysl/2121.15 11.571 68.7879 1.401827 0.56779 Val 117.15 8.673 51.5593 1.0160420.440116 Met 149.21 6.405 38.0762 0.95569 0.255189 ILe 131.17 13.6280.9687 1.786535 0.617281 Total 168.213 1000 28.1433 nm/mlHyp=hydroxy-proline; HyLys=hydroxy-lysine; Cys1/2=Cystine

EXAMPLE 7 Peptide Screening by FACS Analysis

Blood is collected (roughly 75 microliters) into 1 ml PBS containing 5μM EDTA and mixed immediately to prevent clotting. The tubes are kept onice. The red blood cells are lysed using either Gey's solution or abuffered ammonium chloride (ACK) solution (or FACS lysis buffer,Bectin-Dickinson). Cells are washed two-three times with FACS buffer(PBS supplemented with either 1% BSA or 5% FBS and containing 0.05%NaN₃). The pellet is suspended from the final wash in roughly 50microliters FACS buffer (or more if more than one analysis is to be doneon a single sample). Roughly 50 microliters of cell suspension is addedto 10 microliters of antibody solution and mixed gently. The properconcentration of antibody to use is determined prior to this step. Thesuspension is placed on ice for roughly 30 minutes. Cells are thenwashed two-three times with FACS buffer and suspended in 200–300microliters of FACS buffer. Cells are incubated (at a ratio of roughly1:100 cells:minicells) with FITC labeled minicells (in PBS at 2 O.D./ml)at room temperature for 15 minutes. (For live/dead discrimination, addroughly 10 microliters propidium iodide (PI) solution (stock solution,10 μg/ml). If cells were to be fixed, PI was not added.

The cells are ready for analysis upon washing two-three times with FACSbuffer and suspended in 200–300 microliters of FACS buffer.

The cells may be alive or fixed at the time of measurement, but are inmonodispersed (single cell) suspension. They are passed single-filethrough a laser beam by continuous flow of a fine stream of thesuspension. Each cell scatters some of the laser light, and also emitsfluorescent light excited by the laser. The cytometer typically measuresseveral parameters simultaneously for each cell (low angle forwardscatter intensity-approximately proportional to cell diameter,orthogonal (90 degree) scatter instensity-approximately proportional tothe quantity of granular structures within the cell, and fluorescenceintensity at several wavelengths).

Light scatter alone is quite useful. It is commonly used to exclude deadcells, cell aggregates, and cell debris from the fluorescence data. Itis sufficient to distinguish lymphocytes from monocytes fromgranulocytes in blood leukocyte samples.

The fluorescence intensity is typically measured at several differentwavelengths simultaneously for each cell. Fluorescent probes are used toreport the quantities of specific components of the cells. Fluorescentantibodies are often used to report the densities of specific surfacereceptors, and thus to distinguish subpopulations of differentiated celltypes, including cells expressing a transgene. By making themfluorescent, the binding of display library to surface receptors can bemeasured. Intracellular components can also be reported by fluorescentprobes, including total DNA/cell (allowing cell cycle analysis),analysis, newly synthesized DNA, specific nucleotide sequences in DNA ormRNA, filamentous actin, and any structure for which an antibody isavailable. Flow cytometry can also monitor rapid changes inintracellular free calcium, membrane potential, pH, or free fatty acids.

Flow cytometers involve fluidics, laser optics, electronic detectors,analog to digital converters, and computers. The optics deliver laserlight focused to a beam a few cell diameters across. The fluidicshydrodynamically focus the cell stream to and within an uncertainty of asmall fraction of a cell diameter, and, in sorters, break the tram intouniform-sized droplets to separate individual cells. The electronicsquantify the faint flashes of scattered and fluorescent light, and,under computer control, electrically charge droplets containing cells ofinterest so that the cell can be deflected into a separate test tube orculture wells. The computer records data for thousands of cells persample, and displays the data graphically.

EXAMPLE 8 Screening Display Library for Peptides that Bind to Stem Cells

Bone marrow from femurs and tibia of mice is prepared by methodsfamiliar to one of ordinary skill in the art. The marrow is flushed andsuspended in 5 mls staining media using a 23 gauge needle and filteredthrough nylon mesh into a 5 ml tube. The cells are pelleted bycentrifugation (300×g) and resuspended in ACK hypotonic lysis solution(red blood cell lysis buffer—0.15 M NH4 Cl, 1 mM KHCO3, 0.1 mM Na2 EDTA,pH 7.3–100 μl/mouse), placed on ice for roughly 5 minutes and washedwith 5 ml HBSS (or PBS plus 2% FCS) and spun. The solution is thenresuspended in a “lineage cocktail” of appropriate antibody dilutionsand buffer as determined by titration. This mixture is then incubated at4° C. on a rotating platform for 30 minutes. To minimize non-specificbinding of lineage antibodies, the mixture is washed and spun twice,firstly through a serum cushion (FCS). The resultant pellet isresuspended in roughly 3 ml of HBSS. DYNABEADS™ are added to a 1:1bead/cell ratio (in 1 ml) and incubated at 4° C. on a rotating platformfor 30 minutes.

At this point large rosettes of cells should be visible by eye followingthe DYNABEAD™ incubation. The mixture is brought to 5 ml with HBSS andplaced on a magnet according to manufacturers specifications. Wash boundbeads, spin, and transfer supernatents to new tube and spin again.Anti-rat IgG PE is added, incubated on ice for 20–30 minutes, and washedtwice. A blocking solution of rat IgG is added (roughly 50 μl), andincubated on ice for roughly 15 minutes. A staining cocktail of Thy1.1,c-Kit, and Sca-1 is used to resuspend the mixture (use roughly 100 μlper mouse and antibody dilutions as determined by titration), andincubate at 4° C. for 30 minutes. The dead and dying cells are labeledwith propidium iodide in staining medium (PI at 1 μg/ml).

This procedure will generally yield 2–5×10⁵ bound peptides. (Average of5000 stem cells/mouse).

EXAMPLE 9 Bioactive Peptides and Functions Derived from Minicell Displayand Activity Assays

The following is a table, Table 2, of bioactive peptides isolated andcharacterized, as described above.

TABLE 2 PEPTIDE SEQUENCE FUNCTIONAL ACTIVITY VLEP (SEQ ID NO:7)Inhibitor of macrophage recruitment by osteopontin, C5a, fibronectinDDDRKWGFC (SEQ ID NO:8) Inhibits cell/collagen interaction DQDQRWGYC(SEQ ID NO:9) Inhibits cell/collagen interaction DRDRAWGYC (SEQ IDNO:1O) Inhibits cell/collagen interaction DRQWGLC (SEQ ID NO:11)Inhibits cell/collagen interaction DADQKFGFC (SEQ ID NO:12) Inhibitscell/collagen interaction ESHQKYGYCGGCDRNNP (SEQ ID NO:13) Inhibitscell/collagen interaction DSVVYGLRSK (SEQ ID NO:14) Inhibits heparinbinding DSVAYGLKSK (SEQ ID NO:15) Inhibits heparin binding DSVAYGLKSRSK(SEQ ID NO:16) Inhibits heparin binding TPVVPTVDTYDGRGD (SEQ ID NO:17)Cell attachment/alpha_(v)beta_(x) specific TPFIPTESANDGRGDSVAW (SEQ IDNO:18) Cell attachment/a1pha_(v)beta_(x) specific CVVVLVL (SEQ ID NO:19)Promotes cell entry of peptides LDSAS (SEQ ID NO:20) Inhibits alpha4integrin binding LDSPPAALS (SEQ ID NO:21) Inhibits alpha4 integrinbinding AADVESPS (SEQ ID NO:22) Inhibits alpha4 integrin bindingWTGGDDSGSPSSPS (SEQ ID NO:23) Inhibits alpha4 integrin binding SDV (SEQID NO:24) Inhibits alpha4 integrin binding EPEESDVGGAADYP (SEQ ID NO:25)Inhibits alpha4 integrin binding QESPSGTDLLVAGSSP (SEQ ID NO:26)Inhibits alpha4 integrin binding TPVVPTVDTYDGRGDSLAY (SEQ ID NO:27) βintegrin binding DKKELAKFQAERSAAS (SEQ ID NO:28) β₃ attachmentHDRKEFAKFEEEERARA (SEQ ID NO:29) β₃ attachment HDRREFAKFQSERSRA (SEQ IDNO:30) β₃ attachment HDRKEVAKFEAERSKA (SEQ ID NO:31) β₃ attachmentQSWKKQGSPSSPQRRSKGGRKP (SEQ ID NO:32) β₃ attachment SDQDNNGKGSHES (SEQID NO:33) Endothelial cell attachment SDQDQDGDGHQDS (SEQ ID NO:34)Endothelial cell attachment GRGDNPS (SEQ ID NO:35) Fibronectin receptorbinding collagenase induction LVPSSKGRGDYLAQSQP (SEQ ID NO:36)Fibronectin receptor binding collagenase induction PNGRGESLAY (SEQ IDNO:37) Inhibits fibroblast attachment, inhibits collagenase inductionDRYLKFRPV (SEQ ID NO:38) Inhibits melanoma cell attachmentHKFVHWKKPVLPSQNNQ (SEQ ID NO:39) Inhibits melanoma cell attachmentKGMNYTVR (SEQ ID NO:40) Inhibits, neutrophils, endothelium,fibrosarcomas melanoma attachment DPGYIGSR (SEQ ID NO:41) Inhibitsendothelial cell attachment VLPTPTPPGYLSSRSSR (SEQ ID NO:42) Inhibitsendothelial cell attachment KNNQKSEPLIGRKKT (SEQ ID NO:43) Inhibits CD44interaction with GAG YYWRQQQKSDPVVSRRRSPS (SEQ ID NO:44) Inhibits CD44interaction with GAG ATWLPPR (SEQ ID NO:45) Anti-angiogenic QVGLKPLV(SEQ ID NO:46) Anti-angiogenic TPTVRGAAGSGNQN (SEQ ID NO:47)Anti-angiogenic HGRFILPWWYAFSPS (SEQ ID NO:48) Inhibit homotypicaggregation of tumor cells KKAKKSRRS (SEQ ID NO:49) Anti-adhesion(cell-cell) KKGKKSKRS (SEQ ID NO:50) Anti-adhesion (cell-cell)RRSRSSTGKKQKSSQSRKTA (SEQ ID NO:51) Anti-adhesion (cell-cell)DGGRGDSLGWYRRGRGGARRSK (SEQ ID NO:52) Apoptotic to tumor cellsAKKAAAKNNQKSEPLIGRKKT KRSR (SEQ ID NO:53) Apoptotic to tumor cells

It is understood that the disclosed invention is no limited to theparticular methodology, protocols, and reagents described as these mayvary. It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are as described. Publications cited herein andthe material for which they are cited are specifically incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying minicell hosts bound to a binding partnercomprising: (a) expressing a fusion protein in a minicell hostcomprising an outer membrane, wherein the fusion protein is encoded by achimeric gene comprising: a DNA fragment encoding an N-terminal fragmentof a 17K antigen of Rickettsia rickettsii consisting essentially of asignal sequence and lipid modification site which mediates localizationof the fusion protein to the outer membrane, and a DNA fragment encodinga second peptide; (b) contacting the minicell host of step (a) with abinding partner; and (c) identifying the minicell hosts bound to thebinding partner.
 2. The method of claim 1 further comprising: (d)isolating the bound or unbound minicell host.
 3. The method of claim 1,wherein the DNA fragment encoding the second peptide is from a DNAlibrary.
 4. The method of claim 1, wherein the binding partner isselected from the group consisting of carbohydrates, sugars, nucleicacid molecules, peptides, proteins, metals, inorganic molecules andsynthetic drugs.
 5. The method of claim 1, wherein the binding partneris selected from the group consisting of receptors, ligands, antibodies,vitamins, cofactors, enzymes, and neuromediators.
 6. The method of claim1 wherein the minicell host is a gram negative bacteria.
 7. The methodof claim 6 wherein the gram negative bacteria is selected from the groupconsisting of E. coli, Salmonella typhimurium, S. anatum, S.enteritidis, S. pullorum, S. senftenberg, S. worthington, Vibriocholera, Erwinia amylovora, and Haemophilus influenzae.
 8. The method ofclaim 1 wherein the minicell host is a gram positive bacteria.
 9. Themethod of claim 8 wherein the gram positive bacteria is Bacillussubtilis.
 10. The method of claim 1 further comprising: (d) isolatingDNA from the gene encoding the fusion protein; and (e) subjecting theisolated DNA to analysis methods selected from the group consisting ofdetermination of DNA base composition, determination of DNA basesequence, determination of molecular weight, and determination ofsecondary structures within the sequence.
 11. The method of claim 1,wherein the DNA fragment encoding the N-terminal fragment of the 17Kantigen comprises the first 213 nucleotides of the open reading frame ofthe 17K antigen of Rickettsia rickettsii.
 12. The method of claim 1,wherein the expression of the fusion protein is controlled by aninducible promoter element.
 13. The method of claim 12 wherein theinducible promoter element is selected from a group consisting of lac,tac, and trp.
 14. The method of claim 1 further comprising: (d) cleavingthe second peptide from the minicell host.
 15. The method of claim 2further comprising: (e) cleaving the second peptide from the minicellhost.
 16. The method of claim 15 further comprising: (f) isolating thepeptide cleaved from the minicell host; and (g) subjecting the isolatedpeptide to methods selected from the group consisting of determinationof amino acid composition, determination of amino acid sequence,determination of isoelectric point, and determination of molecularweight.
 17. The method of claim 3, wherein the DNA fragment encoding thesecond peptide is at least three amino acids in length.
 18. The methodof claim 11 wherein the DNA fragment encoding the N-terminal fragment ofthe 17K antigen consists of the nucleic acid sequence of SEQ ID NO: 6.19. The method of claim 1, wherein the N-terminal fragment of the 17Kantigen comprises the first 71 amino acids of the 17K antigen.
 20. Themethod of claim 19, wherein the N-terminal fragment of the 17K antigenconsists of the amino acid sequence of SEQ ID NO: 5.